Project Summary/Abstract Autophagy is an essential catabolic process that cells rely on to breakdown cytoplasmic constituents. In-vitro and pre-clinical animal studies indicate a pro-tumorigenic role for autophagy, leading to the launch of over 60 clinical trials utilizing autophagy inhibition. Initial results from clinical trials have been promising, however, these studies already indicate both inherent and acquired resistance to autophagy inhibition in certain tumors. I hypothesize that understanding how tumors circumvent autophagy inhibition will improve the use of autophagy-targeted therapeutics. In my recently published studies, I created a dynamic, Live-Cell CRISPR/Cas9 assay (LC-CRISPR) to target 12 core autophagy genes in a panel of 8 cancer cell lines. I identified autophagy dependent cell lines, however, I also discovered that, with enough selective pressure, even highly autophagy dependent cells can survive loss of autophagy ? specifically loss of the core autophagy proteins ATG7 and FIP200. I discovered that the newly-acquired, autophagy-independent, ATG7-/- and FIP200-/- cells acquired an increased dependence on NRF2 cytoprotective and antioxidant signaling to maintain protein homeostasis. My initial studies also suggest the ATG7-/- and FIP200-/- cells have increased additional cytoprotective processes such as mitochondrial fusion and mitochondrial derived vesicles (MDVs). Previous reports have linked autophagy and NRF2 signaling, however, this will be the first study to understand the role of NRF2 in dictating autophagy dependence. These studies will utilize novel techniques that I have developed including LC-CRISPR and rapid optogenetic inhibition of autophagy to understand the necessity and sufficiency, as well as the in-depth mechanism, rate, and duration of NRF2 antioxidant signaling in mediating autophagy dependence in cancer. I hypothesize that NRF2 expression, both basal and induced expression after autophagy inhibition will dictate autophagy dependence in cancer cells. The 2nd aim and 3rd aims will utilize the isogeneic autophagy dependent WT and ATG7-/- and FIP200-/- autophagy-independent cell lines as well as Atg5-/- mouse tumor cells that circumvented autophagy inhibition in a live animal to understand the links between autophagy dependence, apoptosis, and mitochondrial dynamics. In a previous study we identified the critical link between autophagy and apoptosis and showed that autophagy inhibition leads to increased FOXO3a binding to PUMA and increases sensitivity to other chemotherapeutic agents. The studies proposed here will test the hypothesis that cells that circumvent autophagy dependence also circumvent the FOXO3a-PUMA mediated link between autophagy and apoptosis due to increased mitochondrial fusion. The final aim will test the hypothesis that MDVs are critical for the removal of damaged portions of mitochondria independent of canonical mitophagy and this activity therefore maintains mitochondrial homeostasis. Together these studies will elucidate how cancer cells can circumvent autophagy dependence and provide insight into mechanisms of resistance to autophagy inhibition. !